Phosphonoalkyl and esterified phosphonoalkyl substituted tetrazole thiols

ABSTRACT

The compounds of this invention are phosphonoalkyl and esterified phosphonoalkyl substituted tetrazole thiols useful for preparing antibacterially active 7-acylamino-3-(phosphonoalkyl and esterified phosphonoalkyl substituted tetrazolylthiomethyl)cephalosporins.

This is a division of application Ser. No. 737,971 filed Nov. 2, 1976now U.S. Pat. No. 4,112,086 issued Sept. 5, 1978.

This invention relates to a new series of cephalosporin compounds whichhave antibacterial activity when administered parenterally and tointermediates for the preparation thereof. In particular, the structuresof the biologically active cephalosporin compounds of this invention arecharacterized by having a phosphonoalkyl or esterified phosphonoalkylsubstituted tetrazolythiomethyl group at the 3-position of the cephemnucleus. Also, this invention extends to methods and compositions fortreating certain bacterial infections using these new compounds as wellas to certain chemical intermediates and methods for preparing thecompounds described hereafter.

The compounds of this invention are represented by the followingstructural formula: ##STR1## in which: W is hydrogen or methoxy;

R¹ is a pharmaceutically acceptable acyl group known as a 7-substituentin the cephalosporin art;

R² and R³ are each hydrogen or alkyl of from one to four carbon atoms;and

n is one to five,

or a non-toxic pharmaceutically acceptable salt or hydrate thereof.

Representative of the 7-acyl substituents of the compounds of formula Iare those selected from the following group: ##STR2## where: X isthienyl, phenyl or phenyl monosubstituted with hydroxy, hydroxymethyl,formamido or ureido;

A is NH₂, OH, COOH, SO₃ H or formyloxy;

Y is cyano, sydnone, pyridone, thienyl, tetrazolyl or aminomethylphenyl;

Z is methyl, trifluoromethyl, trifluoroethyl, pyridyl or cyanomethyl;and

m is zero to two.

A group of compounds of this invention is represented by Formula I wheren is one.

Another group of compounds of this invention comprises those compoundsof Formula I where n is one and R² and R³ are hydrogen.

Yet another group consists of the compounds of Formula I where n is one,one of R² and R³ is alkyl and the other is hydrogen.

Still another group consists of the compounds of Formula I where n isone and R² and R³ are both alkyl.

A selected group of the compounds of Formula I are those where n is one,R² and R³ are each ethyl or hydrogen, W is hydrogen, R¹ is ##STR3## X isphenyl or hydroxyphenyl and A is NH₂ or OH.

Some examples of the 7-acyl substituents (R¹ NH--) of the compounds ofFormula I are listed below:

α-hydroxyphenylacetamido

α-aminophenylacetamido

α-amino-4-hydroxyphenylacetamido

trifluoromethylthioacetamido

2,2,2-trifluoroethylsulfinylacetamido

2,2,2-trifluoroethylthioacetamido

cyanoacetamido

α-carboxythienylacetamido

α-carboxyphenylacetamido

α-sulfophenylacetamido

methylsulfonylacetamido

cyanomethylthioacetamido

3-sydnoneacetamido

1-tetrazolylacetamido

2-thienylacetamido

4-pyridylthioacetamido

2-aminomethylphenylacetamido.

Some examples of the compounds of this invention are7β-D-mandelamino-3-(1-diethoxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid,7β-D-mandelamido-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid,7β-D-mandelamido-3-(1-phosphonomethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid,7β-(2-thienylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyl-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid,7β-(1-tetrazolylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid,7β-cyanomethylthioacetamido-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid,7β-(D-α-aminophenylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid,7β-(D-α-amino-4-hydroxyphenylacetamido)-3-(1-ethoxyhydroxyphosphinyl-methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid,7β-D-mandelamino-7α-methoxy-3-(1-phosphonomethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid,7β-(D-α-aminophenylacetamido)-7α-methoxy-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid,7α-methoxy-7β-trifluoromethylthioacetamido-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid,7α-methoxy-7β-(2-thienylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid,7β-D-mandelamino-7α-methoxy-3-(1-diethoxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid and7β-D-mandelamido-7α-methoxy-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

It will be recognized that the 4-carboxylic acid group of the compoundsof Formula I may be readily esterified by methods well known to the art.These esters include, for example, simple alkyl and aryl esters as wellas esters which are easily cleaved, within the body, to the parent acidsuc as indanyl, pivaloyloxymethyl, acetoxymethyl, propionyloxymethyl,glycyloxymethyl, phenylglycyloxymethyl and thienylglycyloxymethyl estersand others. Of course, when A is COOH, this group may be similarlyesterified. All such esters are included within the scope of thisinvention.

Cephalosporin derivatives having 7-acyl substituents as defined aboveare well documented in the prior art. Substitution by variouslysubstituted S-heterocyclicthiomethyl-groups, includingtetrazolylthiomethyl, at the 3-position of the cephem nucleus is alsoknown. No references to cephalosporin compounds contaning the3-(phosphonoalkyl or esterified phosphonoalkyl substitutedtetrazolyl)thiomethyl moiety disclosed herein are believed to be knownto the art.

When W is hydrogen, the compounds of Formula I are preferably preparedby acylating 7β-aminocephalosporanic acid (7-ACA) with an appropriateacylating agent, suitably protected as necessary, and then displacingthe 3-acetoxy group with the desired phosphonoalkyl or esterifiedphosphonoalkyltetrazole thiol of the formula: ##STR4## in which: n isone to five; and

R² and R³ are each hydrogen or alkyl of from one to four carbon atoms;

with subsequent removal of the protective group(s). When certainacylating agents are used, for example activated and protectedderivatives of mandelic acid, it is preferable to remove the protectinggroup from the 7-sidechain prior to displacement.

The carboxylic acid group of the acylating agent in the first step ofthis reaction, the 7-acylation, is activated by any of the standardmethods such as conversion to the mixed anhydride, acid chloride, acidimidazolide or activated ester. In addition, a reagent such asdicyclohexylcarbodimide can be used provided that the carboxyl group onthe cephem nucleus is protected with an easily removable protectinggroup such as a benzhydryl, t-butyl, trichloroethyl, benzyl,benzyloxymethyl, p-methoxybenzyl or p-nitrobenzyl ester. When A is NH₂,the α-amino group of the acylating agent is, preferably, protected priorto acylation with an easily removable protective group known in the artsuch as t-butoxycarbonyl, trichloroethoxycarbonyl, benzyloxycarbonyl,the method acetoacetate adduct or similar groups commonly used in thesynthesis of peptides.

Alternatively, and preferably when W is methoxy, the compounds ofFormula I are prepared by acylation as described above, of anappropriate 7β-amino-3-(phosphonoalkyl or esterified phosphonoalkylsubstituted tetrazolylthiomethyl)cephalosporin nucleus of Formula III:##STR5## in which: W is hydrogen or methoxy;

n is one to five;

R² and R³ are each hydrogen or alkyl of from one to four carbon atoms;and

R⁴ is hydrogen or a protecting group, with an appropriate acylatingagent followed by removal of the protective groups when present.

The protective groups can be removed according to methods well known tothe art, such as with trifluoroacetic acid when t-butyl ort-butoxycarbonyl protective groups are used. The resulting salt isconverted to the zwitterionic product or to the free acid by means of abasic ion exchange resin such as polystyrene-amine ion exchange resin(for example, Amberlite IR-45) or else by basification of an aqueoussolution of the salt.

The acylating agents used as starting materials are either known orprepared by known methods.

The 7β-amino-3-(phosphonoalkyl and esterifiedphosphonoalkyltetrazolylthiomethyl)cephalosporin starting materials ofFormula III where W is hydrogen are prepared by reaction of7β-aminocephalosporanic acid and a substituted tetrazole thiol ofFormula II and then esterifying.

When W is methoxy, the 7β-amino-7α-methoxy cephalosporin nuclei ofFormula III are prepared by reaction of the corresponding7β-amino-3-(substituted tetrazolylthiomethyl)-cephalosporin where W ishydrogen and R⁴ is a protecting group such as a t-butyl group with3,5-di-t-butyl-4-hydroxybenzaldehyde with azeotropic removal of water.Subsequent treatment of the product thus formed with lead dioxide andreaction of the oxidized intermediate with methanol followed by cleavageof the imine function with, for example, Girard reagent T(trimethylaminoacetohydrazide chloride), followed by removal of theprotective group(s) as desired gives the compounds of Formula III. Wheneither or both of R² and R³ are hydrogen, the free hydroxyl group(s) isalso protected, for example as a t-butyl ester.

The dialkoxyphosphinylalkyltetrazole thiols of Formula II where R² andR³ are both alkyl are prepared by reaction of adialkylphosphinylalkyldithiocarbamate such as methyl1-(diethoxyphosphinyl)methyldithiocarbamate with an azide such as sodiumazide. The dialkoxyphosphinylalkyldithiocarbamates are prepared bytreatment of a dialkyl aminoalkylphosphonic acid, for example diethylaminomethylphosphonate, with carbon disulfide and an alkyl halide suchas methyl iodide in the presence of a base such as potassium hydroxide.

The aminoalkylphosphonate dialkyl esters not known to the art areprepared by treatment of a dialkyl phthalimidoalkylphosphonate withhydrazine according to the procedure of Yamauchi et al., Bull. Chem.Soc. Japan 48:3285 (1975). The dialkyl phthalimidoalkylphosphonates areprepared via reaction of a N-hydroxyalkylphthalimide with phosphorustribromide followed by reaction of the N-bromoalkylphthalimide thusformed with a trialkylphosphite as described by Yamauchi et al., Bull.Chem. Soc. Japan 45:2531 (1972).

When one of R² and R³ is hydrogen and the other is alkyl, thealkoxyhydroxyphosphinylalkyltetrazole thiols of Formula II are preparedby basic hydrolysis of the correspondingdialkoxyphosphinylalkyltetrazole thiols.

When R² and R³ are both hydrogen, the phosphonoalkyltetrazole thiols ofFormula II are prepared by treatment of the correspondingdialkoxyphosphinylalkyltetrazole thiols with a mixture of concentratedhydrochloric and acetic acids.

The compounds of Formulas II and III are also considered as objects ofthis invention.

Certain compounds of this invention are capable of forming salts with,for example, the alkali metals such as sodium or potassium, the alkalineearth metals such as calcium or with the ammonium cation. When A is NH₂,the compounds can exist as the zwitterion or as either an acid or basesalt. These salts are prepared by standard methods using a wide varietyof non-toxic pharmaceutically acceptable acids and bases known in theart and are also considered as objects of this invention.

The compounds of Formula I and salts thereof may also exist as hydratesor solvates. All such hydrates, solvates and fractions thereof areconsidered as being encompassed within the scope of this invention.

It will be recognized that due to the asymmetric α-carbon atom in the7-acetamido group of Formula I where R¹ is ##STR6## optical isomers willexist. Racemic or resolved products are obtained depending upon whethera racemic or resolved sidechain acid is used as an acylating agent. Theresolved sidechain acids are readily obtained from the racemic compoundsby resolution according to well known methods, including fractionalcrystallization of a salt formed with an optically active acid or base.All of the isomers, including separated isomers and mixtures thereof,are included within the scope of this invention.

The compounds of Formula I have anti-bacterial activity against bothGram-positive and Gram-negative organisms. Minimum inhibitoryconcentrations (MIC's) range from 0.1 to >200 μg/ml in in vitro testing.Test results for the compounds7β-D-mandelamido-3-(1-diethoxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid sodium salt hydrate (Compound A),7β-D-mandelamido-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid disodium salt hydrate (Compound B) and7β-D-mandelamido-3-(1-phosphonomethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid (Compound C) are given below. Results shown in parenthesis arethose obtained for cefazolin.

    ______________________________________                                                  MIC(μg/ml)                                                       Bacteria    Compound A Compound B Compound C                                  ______________________________________                                        S. aureus   3.1, 6.3 (0.4)                                                                           6.3 (0.4)  6.3 (0.4)                                   SK 127                                                                        S. aureus   0.8 (0.4)  1.6 (0.2)  3.1 (0.2)                                   SK 23390                                                                      S. villaluz 50 (200)   >200 (100) >200 (200)                                  SK 70390                                                                      Strep. faecalis                                                                           12.5, 25 (6.3)                                                                           100, 50 (6.3)                                                                            200 (6.3)                                   HH 34358                                                                      E. coli     6.3 (0.8)  0.4, 0.8 (0.8)                                                                           3.1 (0.8)                                   SK 12140                                                                      E. coli     12.5 (1.6),                                                                              1.6 (0.8)  3.1 (0.8)                                   HH 33779    6.3 (0.8)                                                         Kleb. pneumo.                                                                             3.1, 1.6 (0.8)                                                                           0.4 (1.6)  1.6 (0.8)                                   SK 4200                                                                       Kleb. pneumo.                                                                             3.1 (1.6)  0.4 (0.8)  0.8 (0.4)                                   SK 1200                                                                       Salmonella  3.1 (0.8), 1.6 (0.8), 0.8 (0.4)                                   ATCC 12176  1.6 (0.4)  0.2 (0.4)                                              Pseudo. aerug.                                                                            >200 (>200)                                                                              >200 (>200)                                                                              >200 (>200)                                 HH 63                                                                         Serratia marc.                                                                            25 (>200), 6.3 (>200),                                                                              3.1 (50)                                    ATCC 13880  12.5 (50)  3.1 (50)                                               Proteus morgani                                                                           3.1 (200), 1.6 (200), 50 (100)                                    179         1.6 (100)  0.1 (100)                                              Entero. aerog.                                                                            12.5 (1.6) 3.1 (1.6)  6.3 (1.6)                                   ATCC 13048                                                                    Entero. cloacae                                                                           3.1 (0.8)  0.8 (0.8)  1.6 (0.8)                                   HH 31254                                                                      Proteus mirabilis                                                                         3.1, 1.6 (3.1)                                                                           1.6 (3.1)  0.4 (3.1)                                   444                                                                           ______________________________________                                    

In the in vivo mouse protection test,7β-D-mandelamido-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid disodium salt hydrate exhibited Ed₅₀ 's at 1.02 mg/kg against E.coli 12140 and 0.39 mg/kg against Kleb. pneumo. 4200 upon subcutaneousinjection; cefazolin gave results of 4.4 mg/kg against E. coli 12140 and7.2 mg/kg against Kleb. pneumo. 4200 upon subcutaneous administration.

Pharmaceutical compositions having antibacterial activity which comprisea pharmaceutical carrier containing an active but non-toxic quantity ofa compound of Formula I as well as methods of combatting bacterialinfections by administering such a composition to an infected host in anon-toxic amount sufficient to combat such infections are also objectsof this invention. The administration may be orally or by parenteralinjection such as subcutaneously, intramuscularly or intravenously. Theinjection of suitably prepared sterile solutions or suspensionscontaining an effective, non-toxic amount of the new cephalosporincompound is the preferred route of administration.

The compounds of Formula I are formulated and administered in the samemanner as other cephalosporins. The dosage regimen comprisesadministration, preferably by injection, of an active but non-toxicquantity of a compound of Formula I selected from the dosage unit rangeof from 100 to 1000 mg with the total daily dosage regimen being from400 to 6 g. The precise dosages are dependent upon the age and weight ofthe subject and on the infection being treated and can be determined bythose skilled in the art based on the data disclosed herein comparedwith that available to the art attained with known cephalosporins.

The following examples illustrate the invention but are not to beconstrued as limiting the scope thereof. Temperatures are in degreesCentigrade (°C.) unless otherwise stated.

EXAMPLE 17β-D-Mandelamido-3-(1-diethoxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

Carbon disulfide (0.88 ml, 1.11 g) was added dropwise to a stirredsolution of 2.45 g (14.6 mmol) of diethyl aminomethylphosphonate and0.82 g (14.6 mmol) of potassium hydroxide in 25 ml of ethanol. Duringaddition the temperature of the reaction mixture rose from 26° to 33°. Asecond portion of 0.88 ml of carbon disulfide was added and the mixturewas stirred at 50° for 1 hour. The mixture was cooled to ambienttemperature, 2.08 g (14.6 mmol) of methyl iodide was added dropwise andthe resulting mixture was stirred for 1.5 hours. The mixture wasevaporated to dryness, the residue was dissolved in water and theaqueous solution was extracted twice with ethyl acetate. The extractswere combined, dried (MgSO₄) and evaporated to dryness to give methyl1-diethoxyphosphinylmethyldithiocarbamate.

To a solution of 2.85 g (11.1 mmol) of methyl1-diethoxyphosphinylmethyldithiocarbamate in 25 ml of ethanol was addeda solution of 0.72 g (11.1 mmol) of sodium azide in 5 ml of water. Thereaction mixture was refluxed for 2.5 hours then evaporated to neardryness. Water (20 ml) was added and the aqueous mixture was layeredwith 40 ml of ethyl acetate and acidified to pH 2.2 by addition of 6 Nsulfuric acid. The layers were separated and the aqueous phase wasextracted twice with ethyl acetate. The ethyl acetate solutions werecombined, dried (MgSO₄) and evaporated to dryness to give1-diethoxyphosphinylmethyltetrazole-5-thiol.

C₆ H₁₃ N₄ O₃ PS

Calculated: 28.57% C; 5.20% H; 22.21% N; Found: 29.39% C; 5.42% H;22.30% N.

To a solution of 3.42 g (8.0 mmol) of 7-D-mandelamidocephalosporanicacid sodium salt in 75 ml of water was added 2.52 g (10.0 mmol) of1-diethoxyphosphinylmethyltetrazole-5-thiol and 0.84 g (10.0 mmol) ofsodium bicarbonate. The reaction mixture was stirred at ca. 70° for 5.5hours while maintaining the pH at 7.2. After cooling to ambienttemperature the mixture was extracted twice with ether. The aqueousphase was layered with ethyl acetate and acidified to pH 1.9. The layerswere separated and the aqueous phase was extracted with ethyl acetate.The ethyl acetate solutions were combined, dried (MgSO₄) and evaporatedto dryness to give a residue which was dissolved in ethyl acetate. Asmall volume of ether was added to the ethyl acetate solution and it wasfiltered. The filtrate was added to 200 ml of rapidly stirring ether andthe solid which formed was collected by filtration, dried in vacuo (P₂O₅) and chromatographed on silica gel with 90:10:3chloroform-ethanol-formic acid is eluant to give the title compound.

A solution of7β-D-mandelamido-3-(1-diethoxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid in methanol was adjusted to pH 6.5 by addition of 5% aqueous sodiummethoxide then filtered. Ether was added to the filtrate and the solidmaterial which formed was collected by filtration to give7β-D-mandelamido-3-(1-diethoxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid sodium salt.

C₂₂ H₂₆ N₆ O₈ PS₂.Na.H₂ O

Calculated: 41.37% C; 4.41% H; 13.16% N; Found: 41.80% C; 4.45% H;12.48% N.

EXAMPLE 27β-D-Mandelamido-3-(1-ethoxyhydroxyphosphinylmethyl-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

A solution of 5.04 g (0.02 mol) of1-diethoxyphosphinylmethyltetrazole-5-thiol in 80 ml of ethanol wastreated with 40 ml (0.04 mol) of 1 N potassium hydroxide solution andthe mixture was refluxed for 1.25 hours. Evaporation of the reactionmixture to dryness gave a residue which was dissolved in 40 ml of 8:2:1chloroform-ethanol-formic acid. The solid which formed was collected anddissolved in 25 ml of water. The aqueous solution was passed through acolumn of Dowex 50W-X8 fonic acid ion-exchange resin to give, afterevaporation of the solvent, a solid material. The solid was dissolved inether and the ether solution was filtered and evaporated to dryness togive 1-ethoxyhydroxyphosphinylmethyltetrazole-5-thiol, m.p. 119.5°-122°.

C₄ H₉ N₄ O₃ PS

Calculated: 21.43% C; 4.08% H; 24.99% N; Found: 21.15% C; 4.04% H;24.77% N.

A mixture of 3.42 g (8.0 mmol) of 7β-D-mandelamidocephalosporanic acidsodium salt, 2.5 g (11.2 mmol) of1-ethoxyhydroxyphosphinylmethyltetrazole-5-thiol and 1.87 g (22.3 mmol)of sodium bicarbonate in 100 ml of water was warmed to 67° and stirredfor 4.75 hours while maintaining the pH at 6.9 by addition of sodiumbicarbonate. The mixture was cooled to ambient temperature, layered with100 ml of ethyl acetate and acidified to pH 1.8 with 6 N sulfuric acid.The layers were separated and the aqueous phase was extracted with ethylacetate. The aqueous layer was brought to pH 7 by addition of aqueoussodium bicarbonate and then chromatographed on XAD-7 resin. The eluatewas concentrated in vacuo, filtered and lyophilized to give the titlecompound as the corresponding disodium salt.

C₂₀ H₂₁ N₆ O₈ PS₂ . 2 Na . 3.5 H₂ O

Calculated: 35.45% C; 4.16% H; 12.40% N; Found: 35.17% C; 3.72% H;12.48% N.

The disodium salt is converted to the title compound by stirring it inaqueous solution with strongly acidic Amberlite IR-120H ion-exchangeresin and then lyophilizing.

EXAMPLE 37β-D-Mandelamido-3-(1-phosphonomethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

A solution of 4.04 g (0.02 mol) of1-diethylphosphinylmethyltetrazole-5-thiol in 100 ml of 1:1 aceticacid-concentrated hydrochloric acid was refluxed for ca. 16 hours. Thesolution was evaporated to give a residue which was chromatographed oncellulose with 90:10 acetonitrile-water as eluant. Theproduct-containing fractions were combined and evaporated to a smallvolume and this solution was passed through a Dowex 50W-X8 sulfonic acidion-exchange resin column to give 1-phosphonomethyltetrazole-5-thiol,m.p. 185°-186° (dec.)

C₂ H₅ N₄ O₃ PS

Calculated: 12.25% C; 2.57% H; 28.57% N; Found: 12.66% C; 2.60% H;28.15% N.

A mixture of 1.92 g (45 mmol) of 7-D-maldelamidocephalosporanic acidsodium salt, 1.2 g (61 mmol) of 1-phosphonomethyltetrazole-5-thiol, 1.01g (120 mmol) of sodium bicarbonate and 50 ml of water was treated withsufficient 5% aqueous sodium bicarbonate solution to give a pH of 6.9and then heated at 67° with stirring for 4 hours. The reaction mixturewas passed through a XAD-7 ion-exchange resin column and theproduct-containing fractions were combined and treated with Dowex 50W-X8sulfonic acid ion-exchange resin in water to bring the pH to 1.4. Theresin was filtered off and the solvents were removed. The residue wasdissolved in 75:25 acetonitrile-water and passed through a column ofmicrocrystalline cellulose. The product-containing fractions werecombined and evaporated to dryness. The residue was dissolved inmethanol and Dowex 50W-X8 resin was added until pH 1.6. The resin wasfiltered off and the remaining solution was cooled in ice and brought topH 7.0 by addition of a 5% solution of sodium methoxide in methanol.Ether was added with stirring and the resulting solid material wascollected by filtration, dissolved in water and lyophilized to give thetitle compound as its disodium salt.

C₁₈ H₁₇ N₆ O₈ PS₂ . 2 Na . 2.5 H₂ O

Calculated: 34.23% C; 3.41% H; 13.30% N; 7.28% Na; Found: 34.65% C;3.52% H; 12.47% N; 7.49% Na.

7β-D-Mandelamido-3-(1-phosphonomethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid disodium salt was converted to the title compound as described inExample 2.

EXAMPLE 47β-(D-α-Aminophenylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

A solution of 7.58 g (0.015 mol) of7β-(D-α-t-butoxycarbonylaminophenylacetamido)cephalosporanic acid, 2.24g (0.01 mol) of 1-ethoxyhydroxyphosphinylmethyltetrazole-5-thiol and2.94 g (0.035 mol) of sodium bicarbonate in 125 ml of water is stirredat 60° for 5 hours while maintaining the pH at 7.0-7.2 by addition ofsodium bicarbonate. The mixture is cooled, acidified to pH 3 with dilutehydrochloric acid and extracted with ethyl acetate. After adjusting thepH back to 7 with sodium bicarbonate, the aqueous phase is passedthrough a XAD-7 resin column and the product-containing fractions arelyophilized to give7β-(D-α-t-butoxycarbonylaminophenylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid sodium salt.

7β-(D-α-t-Butoxycarbonylaminophenylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid sodium salt (ca. 1 g) is stirred at 25° with 25 ml oftrifluoroacetic acid and 25 ml of 1,3-dimethoxybenzene for 2.25 hours.The mixture is evaporated to dryness in vacuo, ether is added to theresidue and the precipitate is collected, washed with ether, stirred inacetonitrile for 2 hours and then collected and dried in vacuo to givethe title compound as its trifluoroacetic acid salt.

An aqueous solution of the trifluoroacetic acid salt is brought to pH5.0 by addition of dilute aqueous sodium hydroxide. Afterlyophilization, the lyophilized material is dissolved in methanol andether is added to precipitate7β-(D-α-aminophenylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid sodium salt. The sodium salt is dissolved in water and the aqueoussolution is passed through an Amberlite IR-120H ion-exchange resincolumn. Lyophilization of the eluted material gives the title compound.

EXAMPLE 5

Reaction of the N-t-butoxycarbonyl derivative of the followingcephalosporanic acids:

7β-(α-amino-4-hydroxyphenylacetamido)cephalosporanic acid

7β-(α-amino-4-formamidophenylacetamido)cephalosporanic acid

7β-(α-amino-3-formamidophenylacetamido)cephalosporanic acid

7β-(α-amino-4-ureidophenylacetamido)cephalosporanic acid

7β-(α-amino-3-ureidophenylacetamido)cephalosporanic acid

7β-(α-amino-4-hydroxymethylphenylacetamido)cephalosporanic acid

with 1-ethoxyhydroxyphosphinylmethyltetrazole-5-thiol as described inthe procedure of Example 4 followed by removal of the protective groupand conversion of the trifluoroacetic acid salt to the free acid asdescribed therein gives the following compounds of this invention:

7β-(α-amino-4-hydroxyphenylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

7β-(α-amino-4-formamidophenylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

7β-(α-amino-3-formamidophenylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

7β-(α-amino-4-ureidophenylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

7β-(α-amino-3-ureidophenylacetamido-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

7β-(α-amino-4-hydroxymethylphenylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

EXAMPLE 6

7β-(4-Hydroxymandelamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid is prepared by reaction of 7β-(4-hydroxymandelamido)cephalosporanicacid sodium salt, 1-ethoxyhydroxyphosphinylmethyltetrazole-5-thiol andsodium bicarbonate as described in the procedure of Example 2 followedby conversion of the product sodium salt to the free acid as describedtherein.

EXAMPLE 7

When the sodium salt of a cephalosporanic acid listed below:

7β-(3-sydnoneacetamido)cephalosporanic acid

7β-(2-thienylacetamido)cephalosporanic acid

7β-(1-tetrazolylacetamido)cephalosporanic acid is reacted with1-ethoxyhydroxyphosphinylmethyltetrazole-5-thiol and sodium bicarbonateby the procedure described in Example 2 and the product is converted tothe free acid as described therein, the following compounds of thisinvention are obtained, respectively:

7β-(3-sydnoneacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

7β-(2-thienylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

7β-(1-tetrazolylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

EXAMPLE 87β-Trifluoromethylthioacetamido-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

A solution of 2.24 g (10.0 mmol) of1-ethoxyhydroxyphosphinylmethyltetrazole-5-thiol, 1.68 g sodiumbicarbonate and 5.45 g (12.5 mmol) of7β-trifluoromethylthioacetamidocephalosporanic acid sodium salt in 60 mlof water is stirred at 70°-75° for 5 hours while maintaining the pH at6.8 by addition of 5% aqueous sodium carbonate solution. The reactionmixture is cooled, acidified to pH 3.0 with dilute hydrochloric acid andextracted with ethyl acetate. After adjusting the pH back to 7 theaqueous phase is passed down an XAD-7 resin column. Theproduct-containing fractions are treated with strongly acidic AmberliteIR-120H ion-exchange resin and then lyophilized to give the titlecompound.

EXAMPLE 9

Reaction of the sodium salt of a cephalosporanic acid listed below:

7β-(2,2,2-trifluoroethylthioacetamido)cephalosporanic acid

7β-trifluoromethylsulfinylacetamidocephalosporanic acid

with 1-ethoxyhydroxyphosphinylmethyltetrazole-5-thiol and sodiumbicarbonate as described in the procedure of Example 8 gives thefollowing compounds of this invention as final products:

7β-(2,2,2-trifluoroethylthioacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

7β-trifluoromethylsulfinylacetamido-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

EXAMPLE 107β-Amino-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

A solution of 13.45 g (0.06 mol) of1-ethoxyhydroxyphosphinylmethyltetrazole-5-thiol, 10.9 g (0.04 mol) of7-aminocephalosporanic acid and 13.4 g (0.16 mol) of sodium bicarbonatein 500 ml water is heated at 65° while maintaining the pH at 7.4-7.6 byaddition of aqueous sodium carbonate solution. After 3 hours thereaction mixture is cooled to ambient temperature and passed through acolumn of XAD-7 resin. The product-containing fractions are lyophilizedand7β-amino-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid disodium salt is collected. The disodium salt is converted to thetitle compound by dissolving in water, adding an amount oftrifluoroacetic acid calculated to convert the salt to the acid form,lyophilizing and triturating the lyophilizate with acetone.

EXAMPLE 11

Reaction of 7-aminocephalosporanic acid with1-diethoxyphosphinylmethyltetrazole-5-thiol and1-phosphonomethyltetrazole-5-thiol in the presence of 1 and 3 molecularequivalents of sodium bicarbonate, respectively, as described in Example10 gives7β-amino-3-(1-diethoxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid and7β-amino-3-(1-phosphonomethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid, respectively.

EXAMPLE 127β-(2,2,2-Trifluoromethylsulfinylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

To a stirred solution of 5.7 g (0.03 mol) of2,2,2-trifluoroethylsulfinylacetic acid and 3.45 g (0.03 mol) ofN-hydroxysuccinimide in 50 ml of tetrahydrofuran at 0° is added 6.2 g(0.031 mol) of dicyclohexylcarbodiimide. The reaction mixture is stirredat 0° for 1 hour then at 25° for 12 hours. The precipitate is filteredand washed with tetrahydrofuran and the filtrate is evaporated todryness to give the activated ester of2,2,2-trifluoroethylsulfinylacetic acid.

A suspension of 4.36 g (0.01 mol) of7β-amino-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid in 50 ml of dry dimethylformamide is treated with 4 ml oftriethylamine and the mixture is stirred for 15 minutes at 25°. A slightexcess of 0.01 mol of the activated ester of2,2,2-trifluoroethylsulfinylacetic acid is added to the mixture and itis stirred an additional hour. The reaction mixture is evaporated todryness and water and ethyl acetate are added to the residue. The layersare separated, the ethyl acetate layer is discarded, fresh ethyl acetateis added to the aqueous phase and it is acidified to pH 2.5 by additionof 6 N hydrochloric acid. The mixture is filtered, the layers areseparated and the aqueous phase is treated with Amberlite IR-120Hion-exchange resin and lyophilized to give the title compound.

EXAMPLE 137β-Methylthioacetamido-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

To a stirred, cooled (-20°) solution of 11.34 g (0.026 mol) of7-amino-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid in 220 ml of 3% sodium bicarbonate and 220 ml of acetone is addeddropwise a solution of 3.66 g (0.029 mol) of methylthioacetyl chloridein 52 ml of acetone, during which time the pH of the reaction mixture ismaintained at 8.0 by addition of 10% sodium hydroxide. After additionthe reaction mixture is stirred an additional 20 minutes at -15°, thenis warmed to 25° and extracted with ether. The aqueous phase is passeddown a XAD-7 resin column and the product-containing fractions arecombined, treated with Amberlite IR-120H ion-exchange resin andlyophilized to yield the title compound.

EXAMPLE 147β-(D-α-Formyloxyphenylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

A mixture of 4.36 g (0.01 mol) of7-amino-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid, 3.97 g (0.02 mol) of the formate ester of D-mandeloyl chloride and5 g of sodium bicarbonate in 100 ml of water and 140 ml of acetone isstirred in the cold for 1 hour, then at 25° for 2 hours. The acetone isevaporated in vacuo and the remaining aqueous mixture is extracted withethyl acetate. The aqueous solution is adjusted to pH 7 and passedthrough a XAD-7 resin column. The product-containing fractions arecombined, treated with IR-120H ion-exchange resin and lyophilized togive the title compound.

EXAMPLE 15

Acylation of7β-amino-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid with an activated derivative of an acid listed below:

cyanoacetic acid

cyanomethylthioacetic acid

4-pyridylthioacetic acid

2-pyridone-N-acetic acid

4-pyridone-N-acetic acid

as described in the procedure of Example 12 gives the followingcompounds of this invention:

7β-cyanoacetamido-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

7β-cyanomethylthioacetamido-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

7β-(4-pyridylthioacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

7β-(2-pyridoneacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

7β-(4-pyridoneacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

EXAMPLE 16

Reaction of a cephalosporanic acid listed below or its correspondingsalt:

7β-(α-hydroxy-2-thienylacetamido)cephalosporanic acid

7β-(α-carboxy-2-thienylacetamido)cephalosporanic acid

7β-(α-sulfophenylacetamido)cephalosporanic acid with1-ethoxyhydroxyphosphinylmethyltetrazol-5-thiol and sodium bicarbonateby procedures described hereinabove gives, after conversion of theproduct to the free acid, the following compounds of this invention:

7β-(α-hydroxy-2-thienylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

7β-(α-carboxy-2-thienylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

7β-(α-sulfophenylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

EXAMPLE 177β-(2,2,2-Trifluoroethylsulfonylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

To a suspension of 21.8 g (0.05 mol) of7β-amino-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid in 500 ml of methylene chloride is added over a 30 minute intervala solution of 60.0 g (0.30 mol) of O-t-butyldiisopropylpseudourea in 100ml of methylene chloride. The mixture is stirred at ambient temperaturefor 72 hours. The precipitate is removed by filtration and the filtrateis evaporated to a residue which is taken up in 200 ml of benzene andfiltered again. The filtrate is extracted with three 100 ml portions ofcold 1 N hydrochloric acid. The aqueous extracts are layered with ethylacetate and the pH is adjusted to 7.5 by addition of solid sodiumbicarbonate. The organic layer is separated and the aqueous phase isextracted with two 150 ml portions of ethyl acetate. The combinedextracts are dried (MgSO₄), filtered and evaporated to dryness to give7β-amino-3-(1-ethoxy-t-butoxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester.

To a solution of 10.42 g (0.019 mol) of7-amino-3-(1-ethoxy-t-butoxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester and 3.9 g (0.019 mol) of2,2,2-trifluoroethylsulfonylacetic acid in tetrahydrofuran is addeddropwise a solution of 3.9 g (0.019 mol) of dicyclohexylcarbodiimide in100 ml of tetrahydrofuran. The reaction mixture is stirred at 25° for 12hours, then filtered and concentrated to about 10 ml. The residue isfiltered and evaporated to dryness to give7β-(2,2,2-trifluoroethylsulfonylacetamido)-3-(1-ethoxy-t-butoxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester.

The ester is dissolved in a 1:3 mixture of 1,3-dimethoxybenzene andtrifluoroacetic acid. The solution is stirred for 3 hours and thenevaporated to dryness. The resulting residue is triturated with ether togive the title compound.

Likewise, 7β-(2,2,2-trifluoroethylsulfonylacetamido)derivatives of theother 7β-amino-3-substituted tetrazole cephalosporins disclosed hereinare prepared.

EXAMPLE 187β-D-Mandelamido-3-[1-(2-diethoxyphosphinylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

A mixture of 5.35 g (0.028 mol) of N-(2-hydroxyethyl)phthalimide and 7.7g (0.028 mol) of phosphorus tribromide is warmed until solution isobtained. The reaction mixture is then cooled and poured into ice water.The resulting solid is collected by filtration and washed with water togive N-(2-bromoethyl)phthalimide.

A mixture of 5.32 g (0.017 mol) of N-(2-bromoethyl)phthalimide and 2.74g (0.017 mol) of triethyl phosphite is heated gently to initiatereaction. After the reaction subsides, heating is continued for 1 hourwith distillation of ethyl bromide. Chloroform is added to the cooledmixture and the solution is washed with water. The organic phase isdried and concentrated to give diethyl phthalimidoethylphosphonate.

A mixture of 4.67 g (0.015 mol) of diethyl phthalimidoethylphosphonatein 30 ml. of ethanol and 1.2 ml of 100% hydrazine hydrate is kept atambient temperature for ca. 16 hours then is refluxed for 2 hours. Themixture is cooled and filtered and the filtrate is concentrated to givediethyl (2-aminoethyl)phosphonate.

Substitution of an equivalent amount of diethyl(2-aminoethyl)phosphonate in place of diethyl aminomethylphosphonate inthe procedure of Example 1 followed by cyclization of thedithiocarbamate thus formed gives1-(2-diethoxyphosphinylethyl)tetrazole-5-thiol.

Reaction of 1-(2-diethoxyphosphinylethyl)tetrazole-5-thiol with7-D-mandelamidocephalosporanic acid sodium salt and sodium bicarbonateas described in Example 1 gives7β-D-mandelamido-3-[1-(2-diethoxyphosphinylethyl)tetrazol-5-ylthiomethyl]-3-cephem-carboxylicacid.

EXAMPLE 19

When N-(3-hydroxypropyl)phthalimide is used as a starting material inplace of N-(2-hydroxyethyl)phthalimide in the procedure of Example 18,1-(3-diethoxyphosphinylpropyl)tetrazole-5-thiol is ultimately prepared.

Similarly, when N-(4-hydroxybutyl)phthalimide is used in place ofN-(2-hydroxyethyl)phthalimide in the procedure of Example 18,1-(4-diethoxyphosphinylbutyl)tetrazole-5-thiol is obtained.

Reaction of 1-(3-diethoxyphosphinylpropyl)tetrazole-5-thiol and1-(4-diethoxyphosphinylbutyl)tetrazole-5-thiol with7-D-mandelamidocephalosporanic acid sodium salt and sodium bicarbonateas described in Example 1 gives7β-D-mandelamido-3-[1-(3-diethoxyphosphinylpropyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid and7β-D-mandelamido-3-[1-(4-diethoxyphosphinylbutyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid, respectively.

EXAMPLE 20

Reaction of N-bromomethylphthalimide with trimethyl phosphite asdescribed in Example 18 for N-(2-bromoethyl)-phthalimide and triethylphosphite gives dimethyl phthalimidomethylphosphonate.

Treatment of dimethyl phthalimidomethylphosphonate with hydrazinehydrate as described in Example 18 followed by substitution of thedimethyl aminomethylphosphonate thus formed in the procedure of Example1 in place of diethyl aminomethylphosphonate and subsequent cyclizationof the product thus formed gives1-dimethoxyphosphinylmethyltetrazole-5-thiol.

Reaction of 1-dimethoxyphosphinylmethyltetrazole-5-thiol with7-D-mandelaminodecephalosporanic acid sodium salt and sodium bicarbonateas described in Example 1 gives7β-D-mandelamido-3-(1-dimethoxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

EXAMPLE 21

Substitution of an equivalent amount of a tetrazole thiol listed below:

1-(2-diethoxyphosphinylethyl)tetrazole-5-thiol

1-(3-diethoxyphosphinylpropyl)tetrazole-5-thiol

1-(4-diethoxyphosphinylbutyl)tetrazole-5-thiol

1-dimethoxyphosphinylmethyltetrazole-5-thiol in the procedure of Example2 in place of 1-diethoxyphosphinylmethyltetrazole-5-thiol gives thefollowing thiol compounds:

1-(2-ethoxyhydroxyphosphinylethyl)tetrazole-5-thiol

1-(3-ethoxyhydroxyphosphinylpropyl)tetrazole-5-thiol

1-(4-ethoxyhydroxyphosphinylbutyl)tetrazole-5-thiol

1-hydroxymethoxyphosphinylmethyltetrazole-5-thiol.

Reaction of an alkoxyhydroxytetrazole thiol listed above with7β-D-mandelaminodocephalosporanic acid sodium salt as described inExample 2 gives the following compounds of this invention:

7β-D-mandelamido-3-[1-(2-ethoxyhydroxyphosphinylethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7β-D-mandelamido-3-[1-(3-ethoxyhydroxyphosphinylpropyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7β-D-mandelamido-3-[1-(4-ethoxyhydroxyphosphinylbutyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7β-D-mandelamido-3-(1-hydroxymethoxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

EXAMPLE 22

When a dialkoxyphosphinylalkyl substituted tetrazole thiol listed inExample 21 is used in the procedure of Example 3 in place of1-diethylphosphinylmethyltetrazole-5-thiol, the following tetrazolethiols are obtained.

1-(2-phosphonoethyl)tetrazole-5-thiol

1-(3-phosphonopropyl)tetrazole-5-thiol

1-(4-phosphonobutyl)tetrazole-5-thiol

1-phosphonomethyltetrazole-5-thiol

Reaction of a phosphonoalkyltetrazole-5-thiol listed above with7β-D-mandelamidocephalosporanic acid sodium salt as described in theprocedure of Example 3 gives the following compounds of this invention:

7β-D-mandelamido-3-[1-(2-phosphonoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7β-D-mandelamido-3-[1-(3-phosphonopropyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7β-D-mandelamido-3-[1-(4-phosphonobutyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7β-D-mandelamido-3-(1-phosphonomethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

EXAMPLE 237β-Cyanoacetamido-7α-methoxy-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

A solution of 1.32 g (2.4 mmol) of7β-amino-3-(1-ethoxy-t-butoxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester and 0.56 g (2.4 mmol) of3,5-di-t-butyl-4-hydroxybenzaldehyde in 100 ml of dry benzene isrefluxed for 4 hours under a Dean-Stark trap. The solution is evaporatedunder reduced pressure to leave a residue which is dissolved in 100 mlof 1,2-dichloroethane and cooled to ca. 5° in an ice bath. Three gramsof freshly prepared lead dioxide is added in portions over 20 minutesuntil the starting material is completely consumed. The mixture isfiltered through Celite and the filter cake is washed with two 20 mlportions of cold 1,2-dichloroethane. The filtrate is treated with 25 mlof methanol (distilled from magnesium) and the reaction mixture isallowed to stand at room temperature until complete consumption of theoxidized intermediate and formation of a new slower-moving product isshown by thin layer chromatography. The mixture is evaporated and theresidue is dissolved in 30 ml of methanol and treated with 2.5 g ofGirard reagent T (trimethylaminoacetohydrazide chloride). The reactionmixture is stirred at room temperature for 3 hours, then evaporated togive a residue which is partitioned between ethyl acetate and 20% sodiumchloride solution. The organic phase is washed with 10% sodium chloridesolution and saturated sodium chloride solution. The organic phase isdried (MgSO₄), filtered and evaporated to dryness to give7β-amino-7α-methoxy-3-(1-ethoxy-t-butoxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester.

A solution of 2.31 g (4 mmol) of7β-amino-7α-methoxy-3-(1-ethoxy-t-butoxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester and 0.60 g (4 mmol) of N,N-diethlaniline in 100 ml ofdry methylene chloride is stirred at 0°-5° while 0.41 g (4 mmol) ofcyanoacetyl chloride in 20 ml of methylene chloride is added over a 10minute period. The mixture is stirred in the cold for 30 minutes andthen at ambient temperature for an additional 30 minutes. The reactionmixture is washed with 100 ml of dilute hydrochloric acid, 100 ml of 5%sodium bicarbonate and water. The organic phase is dried and evaporatedto give a residue which is dissolved in 20 ml of 2:1 trifluoroaceticacid-m-dimethoxybenzene and stirred for 3 hours. Excess trifluoroaceticacid is evaporated and the residue is added to 200 ml of rapidly stirredether. The resulting precipitate is collected, washed well with etherand dried to give the title compound.

EXAMPLE 247β-(D-α-aminophenylacetamido)-7α-methoxy-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

To a solution of 2.31 g (4 mmol) of7β-amino-7α-methoxy-3-(1-ethoxy-t-butoxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester and 1.00 g (4 mmol) ofD-α-(N-t-butoxycarbonyl)phenylglycine in 50 ml of dry tetrahydrofuran isadded 0.82 g (4 mmol) of dicyclohexylcarbodiimide. The mixture isstirred at ambient temperature for 3 hours. The precipitated urea isremoved by filtration and the solvent is evaporated to leave a residuewhich is taken up in 100 ml of chloroform and washed with 100 mlportions of dilute hydrochloric acid, 5% aqueous sodium bicarbonate andwater. The organic layer is separated, dried and evaporated to give aresidue which is dissolved in 20 ml of 2:1 trifluoroaceticacid-m-dimethoxybenzene and stirred for 3 hours. Excess trifluoroaceticacid is evaporated under vacuum and the residue is added dropwise to 300ml of rapidly stirred ether. The precipitate is removed by filtration,washed with ether and dried to give the title compound as itstrifluoroacetic acid salt.

An aqueous solution of the trifluoroacetic acid salt is treated with 1molecular equivalent of sodium bicarbonate and then lyophilized. Thelyophilizate is triturated with acetone to give the title compound.

EXAMPLE 257β-D-Mandelamido-7α-methoxy-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

A solution of 1.16 g (2 mmol) of7β-amino-7β-methoxy-3-(1-ethoxy-t-butoxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid t-butyl ester and 0.30 g (2 mmol) of N,N-diethylaniline in 100 mlof dry methylene chloride is stirred at 0°-5° while 0.56 g (2 mmol) ofD-O-dichloroacetylmandeloyl chloride in 10 ml of methylene chloride isadded dropwise over 10 minutes. The mixture is stirred in the cold for30 minutes then warmed to room temperature and stirred for an additional30 minutes. The solution is washed with 50 ml of cold dilutehydrochloric acid and 50 ml of cold 5% aqueous sodium bicarbonate, driedand evaporated to dryness. The residue is dissolved in a mixture of 10ml of trifluoroacetic acid and 2 ml of m-dimethoxybenzene and stirred atambient temperature for 2 hours. The excess trifluoroacetic acid isevaporated under vaccum and the residue is partitioned between 50 ml ofether and 50 ml of water and adjusted to pH 9.3-9.5 with 5% aqueoussodium carbonate. The organic phase is separated and discarded. Theaqueous phase is stirred at pH 9.3-9.5 for 30 minutes, layered withethyl acetate and adjusted to pH 3.0 with dilute hydrochloric acid.After separation of the layers, the aqueous phase is adjusted to pH 7with sodium bicarbonate and passed through a XAD-7 resin column. Theproduct-containing fractions are combined, stirred with AmberliteIR-120H ion-exchange resin and lyophilized to give the title compound.

EXAMPLE 267α-Methoxy-7β-(2-thienylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

A solution of 1.28 g (3 mmol) of7α-methoxy-7β-(2-thienylacetamido)cephalosporanic acid sodium salt isdissolved in 50 ml of water.1-Ethoxyhydroxyphosphinylmethyltetrazole-5-thiol (1.12 g, 4.5 mmol) andsufficient sodium bicarbonate to bring the pH to 6.8 are added and thesolution is heated at 70° until thin layer chromatography indicatesconsumption of the starting cephalosporanic acid (ca. 5 hours). Aftercooling to ambient temperature, the reaction mixture is passed through aXAD-7 resin column and the product-containing fractions are combined,stirred with Amberlite IR-120H ion-exchange resin and lyophilized togive the title compound.

EXAMPLE 277α-Methoxy-7β-trifluoromethylthioacetamido-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

To a cold solution of 5.25 g (0.012 mol) of7β-amino-7α-methoxycephalosporanic acid benzhydryl ester in 200 ml ofmethylene chloride containing 1.79 g (0.012 mol) of N,N-diethylanilineis added dropwise over a 20 minute period a solution of 1.82 g (0.012mol) of trifluoromethylthioacetyl chloride in 50 ml of methylenechloride. After stirring for 30 minutes, the mixture is extractedsuccessively with 5% aqueous sodium bicarbonate, 5% aqueous hydrochloricacid and finally with brine. The organic phase is dried (MgSO₄) and thesolvent evaporated to give7α-methoxy-7β-trifluoromethylthioacetamidocephalosporanic acidbenzhydryl ester.

7α-Methoxy-7β-trifluoromethylthioacetamidocephalosporanic acidbenzhydryl ester is dissolved in a cold mixture of trifluoroaceticacid-anisole (2:1) and the mixture is stirred for 1.5 hours withoutexternal cooling. The solvent is evaporated in vacuo and the residualproduct is taken up in ethyl acetate, washed with water, dried (MgSO₄)and concentrated in vacuo to a small volume. This solution is addeddropwise to stirred petroleum ether to yield7α-methoxy-7β-trifluoromethylthioacetamidocephalosporanic acid.

7α-Methoxy-7β-trifluoromethylthioacetamidocephalosporanic acid (2.2 g, 5mmol) is suspended in 75 ml of water and 0.4 g of solid sodiumbicarbonate is added. To this solution is added 1.7 g (7.5 mmol) of1-ethoxyhydroxyphosphinylmethyltetrazole-5-thiol and sufficient sodiumbicarbonate to bring the pH to 7.5. The mixture is heated at 70° for 7hours while maintaining the pH at 7.5. Progress of the reaction ismonitored by thin layer chromatography and judged to be complete whentlc indicates disappearance of starting material (ca. 7 hours). Thereaction mixture is then cooled to ambient temperature and theproduct-containing fractions are combined, stirred with AmberliteIR-120H ion-exchange resin and lyophilized to give the title compound.

EXAMPLE 287α-(2-Aminomethylphenylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

To a stirred solution of 2.65 g (0.01 mol) of2-(N-t-butoxycarbonylaminomethyl)phenylacetic acid in 75 ml of drytetrahydrofuran at ca. -10° was added dropwise 1.36 g (0.01 mol) ofisobutychloroformate and 2 drops of methyl morpholine. The reactionmixture was stirred at -10° for 10 minutes then a cooled (0°) solutionof 2.72 g (0.01 mol) of 7β-aminocephalosporanic acid in 50 ml of 50%tetrahydrofuran containing 1.01 g of triethylamine was added dropwisewhile maintaining the temperature at ca. -10°. The mixture was stirredat -15° for 1 hour, then at ambient temperature for 2 hours. The pH wasadjusted to 7.5 with sodium bicarbonate and the mixture was extractedrepeatedly with ethyl acetate. The extracts were combined, filteredthrough Celite, dried (MgSO₄) and evaporated to dryness to give7β-(2-N-t-butoxycarbonylaminomethylphenylacetamino)cephalosporanic acid.

7β-(2-N-t-Butoxycarbonylaminomethylphenylacetamido)-cephalosporanic acid(7.79 g, 0.015 mol) and 2.24 g (0.01 mol) of1-ethoxyhydroxyphosphinylmethyltetrazole-5-thiol are suspended in 100 mlof water and sufficient sodium bicarbonate is added to give a solutionof pH 7.0. This solution is heated at 60° for 4 hours, cooled to ambienttemperature and acidified to pH 3.0 with dilute hydrochloric acid. Afterextracting with ethyl acetate, the aqueous solution is sadjusted to pH7.0 by addition of sodium bicarbonate and passed through a XAD-7 resincolumn. The product-containing fractions are lyophilized to give7β-(2-N-t-butoxycarbonylaminomethylphenylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid disodium salt.

7β-(2-N-t-Butoxycarbonylaminomethylphenylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid disodium salt (ca. 1 g) is stirred at 25° with 25 ml oftrifluoroacetic acid and 25 ml of 1,3-dimethoxybenzene for 2.25 hours.The mixture is evaporated to dryness in vacuo, ether is added to theresidue and the precipitate is collected, washed with ether, stirred inacetonitrile for 2 hours and then collected and dried in vacuo to givethe title compound as its trifluoroacetic acid salt.

An aqueous solution of the trifluoroacetic acid salt is brought to pH5.0 by addition of dilute aqueous sodium hydroxide. Afterlyophilization, the lyophilized material is dissolved in methanol andether is added to precipitate7β-(2-aminomethylphenylacetamido)-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid sodium salt. The sodium salt is dissolved in water and the aqueoussolution is passed through an Amberlite IR-120H ion-exchange resincolumn. Lyophilization of the eluted material gives the title compound.

EXAMPLE 29

An injectable pharmaceutical composition is formed by adding sterilewater or sterile saline solution (2 ml) to 500 mg of7β-D-mandelamido-3-(1-ethoxyhydroxyphosphinylmethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid disodium salt. A unit dose is administered intramuscularly to asubject infected with an organism susceptible to the compound as notedherebefore every 4 to 6 hours. Intravenous or drip administration isalso similary used.

Similarly, pharmaceutical compositions of the other compounds of thisinvention may be prepared. What is claimed is:

1. A compound of the formula: ##STR7## in which: n is one to five; andR²and R³ are each hydrogen or alkyl of from one tofour carbon atoms.
 2. Acompound according to claim 1 in which n is one.
 3. A compound accordingto claim 2 in which R² and R³ are hydrogen.
 4. A compound according toclaim 2 in which one of R² and R³ is alkyl and the other is hydrogen. 5.A compound according to claim 2 in which R² and R³ are alkyl.
 6. Acompound according to claim 4, said compound being1-ethoxyhydroxyphosphinylmethyltetrazole-5-thiol.
 7. A compoundaccording to claim 5, said compound being1-diethoxyphosphinylmethyltetrazole-5-thiol.